Synthesis and biochemical analysis of complex chain-elongation intermediates for interrogation of molecular specificity in the erythromycin and pikromycin polyketide synthases

Article abstract of DOI:10.1021/ja9060596

The 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) polyketide synthase (PKS) are unique multifunctional enzyme systems that are responsible for the biosynthesis of the erythromycin and pikromycin 14-membered ring aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing, and macrocyclization. In these studies, the native pentaketide intermediate for DEBS was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in engineered monomodules Ery5, Ery5-TE, and Ery6. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles and suggest that substrate recognition has evolved unique characteristics in each system.

Full text of DOI:10.1021/ja9060596

Published on Web 10/07/2009
Synthesis and Biochemical Analysis of Complex
Chain-Elongation Intermediates for Interrogation of Molecular
Specificity in the Erythromycin and Pikromycin Polyketide
Synthases
Jonathan D. Mortison, Jeffrey D. Kittendorf, and David H. Sherman*
Life Sciences Institute, Department of Chemistry and Department of Medicinal Chemistry,
210 Washtenaw AVenue, UniVersity of Michigan, Ann Arbor, Michigan 48109
Received July 20, 2009; E-mail: davidhs@umich.edu
Abstract: The 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) polyketide synthase (PKS)
are unique multifunctional enzyme systems that are responsible for the biosynthesis of the erythromycin
and pikromycin 14-membered ring aglycones, respectively. Together, these natural product biosynthetic
systems provide excellent platforms to examine the fundamental structural and catalytic elements that
govern polyketide assembly, processing, and macrocyclization. In these studies, the native pentaketide
intermediate for DEBS was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone
products in engineered monomodules Ery5, Ery5-TE, and Ery6. A comparative analysis was performed
with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), dissecting key similarities and
differences between these highly related PKSs. The data revealed that individual modules in the DEBS
and Pik PKSs possess distinctive molecular selectivity profiles and suggest that substrate recognition has
evolved unique characteristics in each system.
Introduction
highlight the need for detailed knowledge regarding the
fundamental mechanistic underpinnings of PKS catalytic
processes.
Biosynthetic assembly of polyketide natural products occurs
on multienzyme megasynthases termed polyketide synthases
(PKSs).1 In these systems, natural product scaffolds are built
from CoA esters of simple organic acid monomers by an
assembly line of enzymatic domains that catalyze their con-
densation into more complex polyketide chains. These domains
are uniquely arranged in bacterial type I PKSs, where they are
organized into covalently bound collinear modules. Each module
is responsible for a single elongation and processing step in
the PKS enzyme pathway.
The unique architecture of type I modular PKS systems
affords the opportunity for rational bioengineering of PKS
enzymes for the generation of novel chemotypes. Predictable
chemical permutations in a polyketide scaffold can be achieved
by a variety of engineering strategies such as the modification
of individual PKS catalytic domains (i.e., inactivation, substitu-
tion, addition, deletion)2,3 or exchange of full modules to form
hybrid PKS systems.4-6 To date, hundreds of novel polyketides
have been produced utilizing these methods. However, in many
cases, engineered PKS proteins have disrupted or significantly
attenuated polyketide production, precluding the efficient gen-
eration of large libraries of new compounds.5-7 These studies
Currently, the most well studied modular type I PKSs are
the 6-deoxyerythronolide B synthase (DEBS, Figure 1)8,9 and
the pikromycin PKS10 (Pik, Figure 2). These two systems share
many similar features, yet contain substantive differences in
modular organization, substrate specificity, and product distribu-
tion. Specific key differences lie in the final two modules (5
and 6) of these PKSs, which are contained on a single bimodular
polypeptide in DEBS to produce one 14-membered ring
macrolactone 6-deoxyerythronolide B (6-DEB), but are on
individual monomodular polypeptides in Pik and produce both
the 12-membered ring macrolactone 10-deoxymethynolide (10-
Dml) and the 14-membered ring macrolactone narbonolide
(Nbl). As a result, the “late” modules of these systems have
been the subjects of significant detailed analysis.
In the DEBS system, many pioneering experiments have
carved out an understanding of modular PKS catalytic efficiency
and substrate specificity. Probing of individual DEBS modules,
including 5 and 6, with short chain diketide model substrates
(6) Menzella, H. G.; Reid, R.; Carney, J. R.; Chandran, S. S.; Reisinger,
S. J.; Patel, K. G.; Hopwood, D. A.; Santi, D. V. Nat. Biotechnol.
2005, 23, 1171–1176.
(1) Staunton, J.; Weissman, K. J. Nat. Prod. Rep. 2001, 18, 380–416.
(2) Donadio, S.; McAlpine, J. B.; Sheldon, P. J.; Jackson, M.; Katz, L.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7119–7123.
(7) McDaniel, R.; Thamchaipenet, A.; Gustafsson, C.; Fu, H.; Betlach,
M.; Betlach, M.; Ashley, G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
1846–1851.
(3) McDaniel, R.; Kao, C. M.; Fu, H.; Hevezi, P.; Gustafsson, C.; Betlach,
M.; Ashley, G.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 1997,
119, 4309–4310.
(8) Cortes, J.; Haydock, S. F.; Roberts, G. A.; Bevitt, D. J.; Leadlay, P. F.
Nature 1990, 348, 176–178.
(9) Donadio, S.; Staver, M. J.; McAlpine, J. B.; Swanson, S. J.; Katz, L.
Science 1991, 252, 675–679.
(4) Tang, L.; Fu, H.; McDaniel, R. Chem. Biol. 2000, 7, 77–84.
(5) Yoon, Y. J.; Beck, B. J.; Kim, B. S.; Kang, H. Y.; Reynolds, K. A.;
Sherman, D. H. Chem. Biol. 2002, 9, 203–214.
(10) Xue, Y.; Zhao, L.; Liu, H. W.; Sherman, D. H. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 12111–12116.
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Erythromycin and Pikromycin Polyketide Synthases
A R T I C L E S
Figure 1. Illustration of the DEBS PKS from the erythromycin biosynthetic pathway. DEBS is responsible for generation of the aglycone 6-deoxyerythronolide
B (6-DEB).
revealed that they each have an inherent molecular selectivity
profile and a high degree of catalytic competence.11-13 However,
the optimal substrates in these in Vitro experiments correlate
poorly to the native substrates that are normally accepted and
processed by these modules in ViVo. This is especially true of
the downstream modules that must accept and process increas-
ingly complex polyketide intermediates in the growing chain.
Comparative study of late modules of the pikromycin PKS14,15
showed that extension and processing of model diketides by
Pik monomodules were nearly 3 orders of magnitude less
efficient than DEBS. More in-depth experiments,16 though,
showed that these diketide model compounds do not effectively
probe the molecular recognition features of these PKS modules.
Rather, utilizing full-length native chain elongation substrates
in biochemical studies of PKS modules yields a more valuable
assessment of kinetic parameters and modular specificity, as
evidenced by the fact that native penta- and hexaketide substrates
for Pik modules 5 (PikAIII) and 6 (PikAIV) were elongated,
processed, and cyclized 2-3 orders of magnitude more ef-
ficiently than the corresponding diketide model substrates.
To further our assessment of the molecular recognition
features in late-stage DEBS modules, we were motivated to
synthesize the native pentaketide substrate and employ it for in
Vitro biochemical analysis of engineered monomodules from
the DEBS3 polypeptide. To establish an effective experimental
system, the bimodular DEBS3 was separated into Ery5 (module
5), Ery5-TE (module 5 + engineered TE fusion), Ery6 (module
6 + native TE fusion), and DEBS TE (excised TE domain).
The catalytic competence and substrate specificity profiles of
these modules were evaluated and compared to corresponding
late monomodules (module 5 and module 6) from the Pik PKS.
(11) Gokhale, R. S.; Tsuji, S. Y.; Cane, D. E.; Khosla, C. Science 1999,
284, 482–485.
(12) Wu, N.; Kudo, F.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2000,
122, 4847–4852.
Results
(13) Wu, N.; Tsuji, S. Y.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2001,
123, 6465–6474.
Total Synthesis of DEBS SNAC Pentaketide Substrate.
Retrosynthetic analysis of the native DEBS pentaketide (1)
revealed an aldol disconnection between C6-C7, to give keto
acid 2 and aldehyde 3 as the required fragments (Scheme 1).
Selectivity for the desired anti-Felkin syn diastereomer could
be achieved in the aldol coupling via a chelation-control
(14) Beck, B. J.; Aldrich, C. C.; Fecik, R. A.; Reynolds, K. A.; Sherman,
D. H. J. Am. Chem. Soc. 2003, 125, 12551–12557.
(15) Yin, Y.; Lu, H.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2003,
125, 5671–5676.
(16) Aldrich, C. C.; Beck, B. J.; Fecik, R. A.; Sherman, D. H. J. Am. Chem.
Soc. 2005, 127, 8441–8452.
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Figure 2. Illustration of the Pik PKS from the methymycin/pikromycin biosynthetic pathway. Pik produces the aglycones 10-deoxymethynolide (10-Dml)
and narbonolide (Nbl).
Scheme 1. Retrosynthetic Analysis
element.17,18 Synthesis of the left fragment started with com-
mercially available (R)-3-bromo-2-methyl-1-propanol, which
was readily converted to iodide 4 by halide displacement and
TBS protection of the primary alcohol (Scheme 2). Diastereo-
selective Myers alkylation19 of 4 with the enolate of (S,S)-
pseudoephedrine propionamide gave amide 5 with high selec-
tivity for the desired 2,4-syn product.20 The amide was then
problematic due to varying degrees of epimerization; however,
one-pot Sharpless oxidation of the TBS ether using RuO4
proved to be a clean and facile route to 2.
21
readily ethylated with EtLi to furnish ketone 6. Originally,
ketone 6 was employed as the intended aldol-coupling partner,
but complications arose in subsequent steps, making the strategy
untenable. This was circumvented by instead using the C1-
oxidized keto acid 2 as the direct coupling partner. Attempts to
oxidize C1 via two-step deprotection/oxidation procedures were
Synthesis of the right fragment 3 began with known Evans
aldol product 7,22,23 which was protected with a benzyl
formacetal group (Scheme 3). The protecting group strategy
applied at the C9 hydroxyl proved crucial for selectivity in the
aldol coupling and for final deprotection of the pentaketide
substrate. Initially, the BOM ether protecting group was utilized
since its extended linker had been thought to allow chelation at
(17) Masamune, S.; Ellingboe, J. W.; Choy, W. J. Am. Chem. Soc. 1982,
104, 5526–5528.
(18) Martin, S. F.; Lee, W. C.; Pacofsky, G. J.; Gist, R. P.; Mulhern, T. A.
J. Am. Chem. Soc. 1994, 116, 4674–4688.
(19) Myers, A. G.; Yang, B. H.; Chen, H.; McKinstry, L.; Kopecky, D. J.;
Gleason, J. L. J. Am. Chem. Soc. 1997, 119, 6496–6511.
(20) Amide 5 was isolated as a single diastereomer. The 2,4-syn diaste-
reomer could be confirmed by reductive removal of the pseudoephed-
rine auxiliary (see ref 19) and TBS deprotection to give the meso 1,5-
diol.
(21) Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K. B. J. Org.
Chem. 1981, 46, 3936–3938.
(22) Crimmins, M. T.; King, B. W.; Tabet, E. A.; Chaudhary, K. J. Org.
Chem. 2001, 66, 894–902.
(23) Pilli, R. A.; de Andrade, C. K. Z.; Souto, C. R. O.; de Meijere, A. J.
Org. Chem. 1998, 63, 7811–7819.
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Erythromycin and Pikromycin Polyketide Synthases
A R T I C L E S
Scheme 2. Synthesis of Left Fragment
Scheme 4. Aldol Coupling and Deprotection
Scheme 3. Synthesis of Right Fragment
oxidative deprotection of the DMBOM group with DDQ, and
deprotection yields suffered somewhat due to competing oxida-
tion to form the DMB orthoester. Nonetheless, this convergent
synthetic strategy provided the target pentaketide SNAC sub-
strate in a sequence of only eight linear steps, avoiding excessive
protecting group and functional group manipulations.
Assay of DEBS Monomodule Activity. Engineered Ery5,
Ery5-TE, and Ery6 were incubated with the synthetic DEBS
pentaketide SNAC substrate and 2-[14C]-methylmalonyl CoA,
and the products were visualized by radio-TLC. In addition,
duplicate reactions were performed utilizing unlabeled meth-
ylmalonyl CoA, and reaction products were analyzed and
confirmed by LC-MS/MS. As demonstrated previously in ViVo,25
the Ery5-TE fusion protein also generates an unnatural 12-
membered ring macrolactone (12) in Vitro (Figures 3 and 4;
Scheme 5). This confirms that DEBS TE is capable of producing
12-membered macrocycles, similar to previous observations
using PikAIII-TE. However, macrolactone 12 occurs with
significant formation of linear hydrolysis products. Interestingly,
in the absence of NADPH cofactor the reaction showed little
production of the predicted 3-oxo macrocyclic derivative (12a)
but instead converted intermediates to an unreduced hexaketide
seco-acid 13. This suggests the possibility of a critical hydrogen-
bonding interaction in the DEBS TE active site involving the
ꢀ-position of the substrate. Thus, in comparison to the Pik TE
where reduction of the enone functionality in the substrate to
the allylic C-7 alcohol abrogated cyclization,26 subtle functional
group changes can also significantly affect the ability of DEBS
TE to catalyze macrocyclization. Also of note was the formation
of some 13 even when NADPH was present in excess with
Ery5-TE. This suggests a kinetic competition between reduction
of the ACP-bound hexaketide by the Ery5 ketoreductase (KR)
domain followed by TE-mediated macrolactonization, or pre-
mature transfer of unreduced hexaketide to the TE active site
prior to reduction resulting in hydrolytic release. Seco-acid 13
was not previously reported from in ViVo studies using an
engineered DEBS1-DEBS2-M5-TE system,25 suggesting that
its formation in Vitro might be caused by suboptimal enzymatic
processing from the unnatural Ery5-TE fusion protein.
the C9 hydroxyl, thus enabling good anti-Felkin selectivity in
analogous aldol couplings.17 However, attempts to remove this
group at the end of the synthesis either failed or resulted in
degradation of the desired pentaketide; therefore, other synthetic
strategies were explored, including new protecting groups for
the C9 hydroxyl. Of the protecting groups surveyed, only related
benzyl formacetals did not abolish aldol selectivity. The
p-methoxybenzyl (PMBOM) formacetal derivative also proved
difficult to remove at the end of the synthesis, but the
3,4-dimethoxybenzyl (DMBOM) derivative was readily depro-
tected. Therefore, Evans-syn aldol product 7 was protected using
DMBOMCl to give 8c, followed by reductive removal of the
Evans auxiliary with LiBH4 to give alcohol 9c. Oxidation to
aldehyde 3c was then best accomplished using Dess-Martin
periodinane.
Coupling of the two fragments proceeded first by enolization
of keto acid 2 with LiHMDS, followed by an aldol reaction
with the DMBOM-protected aldehyde 3c to give the full carbon
chain of the DEBS pentaketide with good (approximately 7:1)
anti-Felkin/Felkin selectivity24 and modest yield (Scheme 4).
This aldol step was followed by subsequent thioesterification
with N-acetyl cysteamine (SNAC). Due to the exceptional acid
sensitivity of 1, buffered conditions were imperative in the final
Next, further probing of the DEBS monomodules was carried
out in Ery5 lacking the terminal thioesterase fusion and the final
module Ery6. Radioassay of Ery5 and Ery6 showed that both
modules appeared to generate low, but detectable, levels of
product when diffusively loaded with the DEBS pentaketide
(Figure S1, Supporting Information) but required further
(24) The anti-Felkin syn diastereomer could be confirmed by conversion
and correlation to a known lactone derivative: Crimmins, M. T.; Slade,
D. J. Org. Lett. 2006, 8, 2191–2194.
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Scheme 5. Illustration of in Vitro Enzymatic Reactions for Cognate
and Non-Cognate Pentaketide/Module 5 Pairs from the Pik and
DEBS PKS
Figure 3. Radio-TLC of reaction products for Ery5-TE incubated with
DEBS pentaketide SNAC. Assay conditions are described in the Experi-
mental Section. Lane 1: No substrate (negative control). Lane 2: No enzyme
(negative control). Lane 3: Ery5-TE w/o NADPH. Lane 4: Ery5-TE w/
NADPH.
Scheme 6. Interrogation of Ery6 with Non-Native DEBS
Pentaketide and Pik Hexaketide (16) Substrates
modules PikAIII and PikAIV) to mediate polyketide chain
transfer between the separated polypeptides. LC-MS/MS analy-
sis confirmed that despite lacking a TE domain, detectable
amounts of unreduced hexaketide seco-acid 13 are released from
Ery5 presumably through adventitious hydrolysis of the inter-
mediate from the Ery5 ACP. Addition to the reaction mixture
of either the DEBS or Pik TE in trans with Ery5, however, did
not restore production of 12. This contrasts with the pikromycin
system where Pik TE is able to function in trans to catalyze
formation of 10-Dml from PikAIII both in ViVo28 and in Vitro.29
Remarkably, we observed that Ery6 was able to accept the
DEBS pentaketide resulting in low levels of macrolactone 12
and hydrolysis product 13 (Scheme 6; Figure 5). This demon-
strates an unexpected flexibility in this module toward incoming
substrates bearing noncognate functionality and chain length.
Cross Reactivity between DEBS and Pik Systems. To probe
the substrate specificity of the DEBS and Pik late modules, both
DEBS pentaketide (1) and Pik pentaketide (14) substrates30 were
assessed for their ability to be loaded, extended, processed, and
cyclized in unnatural substrate/PKS combinations (e.g., DEBS
pentaketide SNAC against PikAIII-TE, Pik pentaketide SNAC
against Ery5-TE) (Scheme 5). Radio-TLC of the reaction
products did not show detectable levels of macrolactones 12 or
10-Dml (15) relative to controls with either PikAIII-TE or Ery5-
TE in reactions containing the respective non-native pentaketide
substrate (Figure 6). LC-MS/MS analysis of the reactions also
did not provide detectable levels of these macrolactones, instead
showing the major products to be the pentaketide seco-acids,
most likely arising from hydrolysis of the parent SNAC
compounds by the terminal thioesterase domains. With PikAIII-
TE, this hydrolytic activity is especially pronounced. PikAIII-
TE incubated with the DEBS pentaketide SNAC revealed low
Figure 4. LC-MS chromatogram in selective ion mode (SIM) of Ery5-TE
incubated with DEBS pentaketide SNAC. Top: Ery5-TE (green). Middle:
Ery5-TE w/o NADPH (red). Bottom: No enzyme control (blue).
structural confirmation to confirm their identity (Vide infra). A
low level or lack of product formation was expected since Ery5
does not have a thioesterase domain to catalyze chain release,
and the pentaketide is not the native substrate for Ery6. Since
the total synthesis of the DEBS hexaketide chain elongation
intermediate proved problematic due to its facile degradation,
Ery6 could not be evaluated with its native substrate at this
time. As expected, the pairing of Ery5-Ery6 did not generate
6-DEB, since the engineered monomodules are now physically
distinct and lack native docking domains27 (unlike in mono-
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Erythromycin and Pikromycin Polyketide Synthases
A R T I C L E S
PikAIII was incubated with the DEBS pentaketide and the
DEBS TE. Neither reaction, however, showed detectable levels
of macrolactones by radio-TLC or LC-MS/MS.
Unlike the experiments involving interrogation of noncognate
pentaketide/module 5 pairs, the module 6 PKSs from Pik and
DEBS demonstrated some flexibility toward non-native sub-
strates. Ery6 was able to accept, extend, process, and cyclize
the non-native Pik hexaketide31 (Scheme 6), though not the Pik
pentaketide. When Ery6 was incubated with Pik hexaketide (16),
both 3-hydroxy-narbonolide (17) and hydrolyzed linear hepta-
ketide (18) were observed in the radio-TLC (Figure S2,
Supporting Information) and LC-MS/MS assay (Figure 5). In
this case, no competition between ketoreduction and cyclization
was detected. Since DEBS hexaketide currently remains un-
available, PikAIV tolerance could not be evaluated in an
analogous experiment. Interestingly, when the DEBS pentaketide
was incubated with PikAIV, detectable levels of hexaketide
seco-acid 13 were generated, though the predicted cyclized
product 12a was absent. This observation conforms to the
current hypothesis regarding ring-formation requirements of the
Pik TE, where a rigid enone at C7-C9 was revealed to be
essential for cyclization of the Pik hexaketide intermediate by
the thioesterase.26 As had been observed previously in Vitro,16
the Pik pentaketide was not accepted by PikAIV to produce
3-oxo-10-Dml.
Finally, bimoduar pairing of PikAIII-PikAIV did not result
in production of predicted macrolactones 12 or 3-oxo-6-DEB
when incubated with the DEBS pentaketide. This is consistent
with the rigid molecular specificity exhibited by PikAIII toward
the DEBS pentaketide. Nonetheless, hydrolyzed hexaketide 13
was observed in this bimodular context (Figure 7), the result of
direct loading, extension, and hydrolysis on the more flexible
PikAIV monomodule.
Figure 5. SIM LC-MS chromatogram of Ery6 reactions. Top: Ery6 w/
DEBS pentaketide SNAC (light blue). Bottom: Ery6 w/ Pik hexaketide
SNAC (purple).
Discussion
Synthesis of the DEBS pentaketide revealed some of the
inherent challenges present in the total synthesis of linear
polyketides, which often suffer from facile degradation path-
ways. This was seen in the final deprotection step of the
pentaketide, where the intermediate readily yielded to acid-
catalyzed hemiketalization and dehydration under most condi-
tions. The synthesis thus required the sparsely used DMBOM
group, which enabled mild deprotection conditions and provided
the necessary compatibility with the earlier aldol step. In
addition, similar degradation pathways thwarted current efforts
toward the desired DEBS hexaketide intermediate. These
challenges highlight an important, yet often underappreciated
aspect of the PKS catalytic mechanism, whereby the linear
polyketide chain elongation intermediates remain covalently
linked to the enzyme complex throughout the PKS extension
process, rather than being diffusively off-loaded after each
catalytic cycle. This mechanism protects the chemically sensitive
Figure 6. Radio-TLC of noncognate pairing of Pik/DEBS pentaketide
SNACs incubated with Ery5-TE and PikAIII-TE. Lane 1: No enzyme w/
Pik pentaketide (negative control). Lane 2: Ery5-TE w/ Pik pentaketide
(No NADPH). Lane 3: Ery5-TE w/ Pik pentaketide. Lane 4: Ery5-TE w/
DEBS pentaketide (positive control). Lane 5: No enzyme w/ DEBS
pentaketide (negative control). Lane 6: PikAIII-TE w/ DEBS pentaketide
(No NADPH). Lane 7: PikAIII-TE w/ DEBS pentaketide. Lane 8: PikAIII-
TE w/ Pik pentaketide (positive control).
(25) Kao, C. M.; Luo, G. L.; Katz, L.; Cane, D. E.; Khosla, C. J. Am.
Chem. Soc. 1995, 117, 9105–9106.
(26) Aldrich, C. C.; Venkatraman, L.; Sherman, D. H.; Fecik, R. A. J. Am.
Chem. Soc. 2005, 127, 8910–8911.
(27) Buchholz, T. J.; Geders, T. W.; Bartley, F. E.; Reynolds, K. A.; Smith,
J. L.; Sherman, D. H. ACS Chem. Biol. 2009, 4, 41–52.
(28) Xue, Y.; Sherman, D. H. Nature 2000, 403, 571–575.
(29) Kittendorf, J. D.; Beck, B. J.; Buchholz, T. J.; Seufert, W.; Sherman,
D. H. Chem. Biol. 2007, 14, 944–954.
levels of hexaketide hydrolysis product 13 by LC-MS/MS,
indicating that some substrate can be accepted and extended
by the enzyme, albeit very inefficiently. To assess the influence
of the TE domain in these cross-PKS assays, noncognate
modules/substrates were incubated together, but native TEs were
added in trans to determine if turnover could be enhanced. Ery5
was thus incubated with Pik pentaketide and the Pik TE, and
(30) The Pik pentaketide was synthesized as previously described. See ref
16.
(31) The Pik hexaketide was synthesized from degradation of 10-DML as
previously described. See ref 16.
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the analogous engineered PikAIII-TE, though this occurred only
when the module was incubated with diketides14 rather than
native substrates. With native Pik pentaketide, no 3-oxo-10-
Dml was observed with PikAIII-TE when NADPH was in-
cluded.16 Thus, unlike Ery5-TE, incomplete reduction in
PikAIII-TE is likely a substrate-driven process rather than a
result of enzyme structural elements that perturb its function.
The differing cyclization activities of the Pik and DEBS TEs
suggest unique substrate binding modes within their active sites.
The oxidation state at the ꢀ-position is important for TE-
mediated cyclization in DEBS, but not in Pik. Thus, it can be
reasoned that the ꢀ-hydroxy group in the extended DEBS
hexaketide acts as a hydrogen-bond donor in the TE active site,
creating an interaction that is vital for efficient cyclization. This
would not be surprising, as the TE has evolved to generate its
sole product 6-DEB from the native heptaketide intermediate,
which also contains a ꢀ-hydroxy group. Abrogation of this
interaction by replacement with a keto group could result in
attenuated cyclization. It has been proposed from modeling of
6-DEB into the active site of the DEBS TE crystal structure
that the 3-position hydroxyl participates in a hydrogen bond
with Asn-180 and the backbone carbonyl of Tyr-171,32 which
is consistent with this observation. This contrasts sharply with
the hypothesized binding mode for Pik TE, which has been
shown from structural studies using affinity-label probes to have
minimal active site hydrogen-bonding interactions with incom-
ing substrates.33 Here, the oxidation state at the ꢀ-position does
not have a critical effect on macrolactonization, as Pik TE
efficiently cyclizes both a hexaketide containing a ꢀ-hydroxy
and a heptaketide containing a ꢀ-keto group to give 10-Dml
and Nbl, respectively.16
While there is evidence that Pik TE has developed direct
protein-protein interactions with PikAIII29 to enable formation
of 10-Dml from this monomodule, Ery5 and the DEBS TE
appear to be unable to engage in a similar type of molecular
recognition. Providing DEBS TE or Pik TE in trans to substrate-
bound Ery5 does not enhance release of macrolactone products.
Similarly, excised DEBS TE provided in trans also failed to
catalyze release of intermediates from PikAIII, strongly sug-
gesting that PikAIII and Pik TE are capable of engaging in a
unique type of molecular interaction.
When PKS substrate specificity is considered, both Pik and
DEBS module 5 appear to have a strong preference for their
native pentaketide chain-elongation intermediates. Both modules
(with engineered TE domains) are capable of efficiently ac-
cepting and processing their respective pentaketide substrates
and cyclizing them to 12-membered ring macrolactones. How-
ever, when incubated with the pentaketide from the reciprocal
system, neither module was tolerant toward the non-native
substrate, despite high structural similarity that differs only at
C6-C7 of the respective polyketide chains. These results
suggest that even small functional changes distal to the enzyme
acyl-thioester linkage are capable of precluding efficient PKS
activity. This contrasts with earlier precursor-directed biosyn-
thesis studies in DEBS, where distal functional changes from
unnatural starter units were well-tolerated by downstream
modules and incorporated into 6-DEB analogues.34 These
modifications at the terminus of the chain elongation intermedi-
Figure 7. SIM LC-MS chromatogram for DEBS pentaketide SNAC
incubated with PikAIII-PikAIV.
and transient intermediates against numerous potential degrada-
tion pathways until they are released as more stable products.
Analysis of the catalytic abilities of the dissected DEBS
monomodules revealed distinctive profiles relative to their
naturally occurring counterparts in the Pik system. From these
studies, the macrocyclization activity of the DEBS and Pik TEs
was revealed to be a key issue, especially in the formation of
12-membered ring products. In Pik, the ability to form 12-
membered macrocycles is well evolved, giving nearly exclusive
formation of 10-Dml or 3-oxo-10-Dml (when NADPH cofactor
is excluded) when presented with the natural hexaketide
substrate. However, with the DEBS TE, the ability to generate
the analogous macrolactones 12 and 12a competes significantly
with hydrolysis activity. This inefficiency appears to stem from
incomplete ketoreduction by the KR of Ery5, followed by
subsequent difficulty in cyclizing the resultant 3-oxo hexaketide
derivative by DEBS TE. It appears that the unnatural TE fusion
facilitates facile cyclization/hydrolysis of unreduced hexaketide
prior to reduction by the KR. The reason for inefficient KR
function is unclear, as it has not been reported in any previous
studies performed in Vitro12,13 or in ViVo25 with DEBS module
5. In this case, the interplay of multiple factors could cause
premature cyclization/hydrolysis of the acyl chain from Ery5-
TE, though structural issues related to the ACP-TE fusion appear
likely to be strong contributors. Since the TE fusion and linker
region are unnatural in this engineered monomodule, this
perturbation might result in premature transfer of extended
hexaketide to the TE active site prior to complete processing
by the ketoreductase. This possibly results from a decrease in
the rate of Ery5 modular catalytic events, an increase in the
rate of intermediate transfer to the thioesterase from the Ery5
ACP, or a combination of both. Similar competition between
reduction and cyclization has also been reported previously with
(32) Tsai, S. C.; Miercke, L. J. W.; Krucinski, J.; Gokhale, R.; Chen,
J. C. H.; Foster, P. G.; Cane, D. E.; Khosla, C.; Stroud, R. M. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 14808–14813.
(33) Akey, D. L.; Kittendorf, J. D.; Giraldes, J. W.; Fecik, R. A.; Sherman,
D. H.; Smith, J. L. Nat. Chem. Biol. 2006, 2, 537–542.
9
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Erythromycin and Pikromycin Polyketide Synthases
A R T I C L E S
ate, however, are located at a position more remote than those
in the substrates from the current studies. Further analysis is
thus required to determine whether the substrate-loading event,
chain-elongation/processing/termination, or all of these steps
represent potential roadblocks to catalysis. Previously, it has
been shown that the PKS channeling mechanism plays a key
role in substrate loading and that diffusive loading of an acyl-
SNAC may not reflect the authentic molecular recognition
features of an individual module. In those studies, inherently
poor diketide model substrates that could not be accepted by
diffusive loading were accepted by PKS modules if presented
as appropriate upstream ACP-bound intermediates.13 Nonethe-
less, these suboptimal substrates were still extended and cyclized
(as triketide latones) at much lower rates than optimized diketide
model compounds. Since these current studies have relied on
diffusive loading, it might be informative to assess the contribu-
tions of channeling to the specificity of the substrate-loading
event with native chain elongation intermediates.
In contrast to Ery5, Ery6 appears to have significantly
increased flexibility toward non-native substrates. Ery6 was
capable of accepting, extending, and cyclizing the noncognate
DEBS pentaketide and Pik hexaketide substrates, though not
the Pik pentaketide intermediate. This was surprising as the
DEBS pentaketide and Pik hexaketide not only have differing
chain lengths (10 and 12 carbons, respectively) but also differ
in their substitution and stereochemistry at the proximal R and
ꢀ positions as well as at more distal positions. The fact that the
Pik pentaketide was not accepted also suggests a key recognition
feature at C6-C7 in Ery6, as these are the only chemically
unique positions compared to DEBS pentaketide. It is possible
that the rigid double bond is structurally untenable for Ery6;
however, the Pik hexaketide also contains an alkene, although
its location is shifted to the C8-C9 position. Thus, since the
presence of the enone alone cannot explain poor activity, the
possibility remains that the position of the enone is a key
consideration. To a lesser degree than Ery6, PikAIV also
demonstrated flexibility in its ability to accommodate the DEBS
pentaketide, catalyzing extension to the hydrolyzed linear
hexaketide seco-acid 13; however, the Pik TE could not cyclize
the pendant DEBS hexaketide intermediate to the expected
macrolactone 12a in this reaction. At the same time, the failure
of PikAIV to accept the Pik pentaketide indicates that this
substrate may not be well tolerated outside of its native module
PikAIII.
architecture, PikAIV also contains an accessible KS active site
and must accept intermediates through an intermodular transfer.
Thus, we might expect that PikAIV would exhibit a similar level
of molecular specificity as PikAIII or Ery5. Nonetheless, PikAIV
is capable of accepting complex non-native intermediates,
though more rigorous interrogation is necessary to fully assess
its specificity.
Based on this comparative analysis, the late modules in the
DEBS and Pik polyketide synthases have evolved unique
specificity profiles and catalytic capacities. Specific insights have
been gained toward understanding key recognition features
necessary for TE-mediated cyclization in these systems, which
is vital for efficient formation of new macrolactone scaffolds
utilizing TE domains as biocatalysts. This new information also
provides an additional basis for future bioengineering efforts
involving a range of PKS modules. Due to their promiscuity,
both Pik and DEBS module 6, especially Ery6, appear to be
excellent candidates for engineering of new biosynthetic path-
ways. Future efforts will seek to assess the kinetic parameters
of the flux through the DEBS modules with native chain-
elongation intermediates, including the contribution of chan-
neling to molecular specificity. Concurrently, continued analysis
of substrate tolerance in modules from both systems will be
assessed using analogues that encompass a variety of stereo-
chemical, structural, and functional group variations.
Experimental Section
Materials and General Procedures. Substrate Synthesis.
Commercially available reagents were purchased from Sigma-
Aldrich, Acros Organics, or Fluka or prepared from established
literature procedures. RuCl3 ·H2O was obtained from Strem Chemi-
cals, Inc. The pikromycin pentaketide and hexaketide SNAC
substrates were prepared as previously described.16 Anhydrous
solvents were obtained either by using an MBRAUN MB-SPS
solvent purification system or by purchasing them in septum-sealed
bottles from Acros Organics (AcroSeal) or EMD (DriSolv).
Diisoproylamine and N,N-diisopropylethylamine were distilled over
CaH2 and stored under argon. Alkyllithiums were titrated prior to
use with diphenylacetic acid. Air- and moisture-sensitive reactions
were carried out under an inert argon atmosphere in flame-dried
glassware. Flash chromatography was carried out using Merck Silica
Gel 60 (230-400 mesh), and thin layer chromatography was
performed on Merck TLC plates precoated with Silica Gel 60 F254
.
1H and 13C NMR spectra were obtained on either a Varian MR400
or an Inova 500 spectrometer. Proton chemical shifts are reported
in ppm relative to TMS with the residual solvent peak as an internal
standard. Proton NMR data are reported as follows: chemical shift
(δ ppm), multiplicity, coupling constant (Hz), and integration. High-
resolution mass spectra were obtained on a Waters Micromass
AutoSpec Ultima Magnetic Sector mass spectrometer.
Taken together, the substrate selectivity profiles that have
emerged from these studies for the late modules from the DEBS
and Pik PKSs suggest rigidity in their fifth modules with
pentaketide substrates, yet tolerance in their sixth and final
module. Module 5 in these PKSs may act as stringent molecular
gatekeepers, especially for diffusive loading of the KS, as
module 5 must mediate intermodular transfer of intermediates
from the upstream module 4, and promiscuous loading of the
exposed KS active site could result in competing enzymatic
pathways or domain inactivation. Module 6, on the other hand,
appears to lack this level of stringency. In the native bimodular
context of DEBS3, the Ery6 KS accepts chain-elongation
intermediates in an intramodular fashion and is likely inacces-
sible to diffusive loading. Thus, increased flexibility would not
be detrimental to overall modular efficiency and substrate
stringency would be unnecessary. Due to its monomodular
Iodide 4. A suspension of (R)-(-)-3-bromo-2-methyl-1-propanol
(4.995 g, 32.64 mmol) and NaI (19.571 g, 130.57 mmol) in 60 mL
of acetone was brought to reflux at 70 °C. After 18 h, the reaction
mixture was diluted with 50 mL of H2O and the volatiles were
removed en Vacuo. The mixture was then extracted with 3 × 50
mL CH2Cl2, and the combined organics were washed with 20 mL
of saturated Na2S2O3, dried over MgSO4, filtered, and concentrated.
The crude iodide was redissolved in 100 mL of CH2Cl2 and
cooled to 0 °C. Imidazole (2.445 g, 35.91 mmol) and TBSCl (5.412
g, 35.91 mmol) were then added sequentially. A white precipitate
formed immediately, and the reaction was monitored by TLC. After
1 h, the reaction was filtered and concentrated. The crude mixture
was resuspended in 100 mL of pentane and filtered, rinsing the
filter with an additional 50 mL of pentane. The filtrate was
concentrated, and the crude oil was purified by flash chromatog-
raphy (100% hexanes) to give 8.358 g (81.5%) of a colorless oil.
(34) Jacobsen, J. R.; Hutchinson, C. R.; Cane, D. E.; Khosla, C. Science
1997, 277, 367–369.
9
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A R T I C L E S
Mortison et al.
1
1H NMR (CDCl3, 399.5 MHz) δ 3.53 (dd, J ) 10.0, 5.0 Hz, 1H),
3.40 (dd, J ) 10.0, 6.9 Hz, 1H), 3.30 (dd, J ) 9.5, 5.2 Hz, 1H),
3.24 (dd, J ) 9.5, 5.6 Hz, 1H), 1.79-1.53 (m, 1H), 0.95 (d, J )
6.7 Hz, 3H), 0.90 (s, 9H), 0.06 (s, 3H), 0.06 (s, 3H); 13C NMR
(CDCl3, 100.5 MHz) δ -5.4, 13.7, 17.2, 18.2, 25.9, 37.4, 66.7;
HRMS CI+ (m/z): 315.0656 (Predicted [M + H]+ for C14H24OSiI
is 315.0641).
0.564 g (82.6%) of a colorless oil. H NMR (CDCl3, 499.9 MHz)
δ 11.86 (br s, 1H), 2.62-2.54 (m, 1H), 2.51-2.37 (m, 3H), 2.03
(ddd, J ) 14.0, 8.9, 6.4 Hz, 1H), 1.32 (ddd, J ) 13.7, 7.7, 5.7 Hz,
1H), 1.14 (d, J ) 7.0 Hz, 3H), 1.05 (d, J ) 7.0 Hz, 3H), 0.98 (t,
J ) 7.3 Hz, 3H); 13C NMR (CDCl3, 125.7 MHz) δ 7.7, 16.5, 17.5,
34.0, 36.1, 37.2, 43.7, 182.5, 214.6; HRMS ESI+ (m/z): 195.1002
(Predicted [M + Na]+ for C9H16O3 is 195.0997).
Oxazolidinone 8c. To a solution of Evans aldol product 7 (2.000
g, 6.87 mmol), TBAI (0.254 g, 0.687 mmol), and DIPEA (5.98
mL, 34.32 mmol) in 50 mL of CH2Cl2 was added DMBOMCl35
(7.437 g, 34.32 mmol) in 20 mL of CH2Cl2 dropwise at 0 °C. The
reaction was warmed to room temperature and stirred for 16 h.
The reaction was quenched with 50 mL of H2O, and the layers
were separated. The aqueous portion was extracted with 2 × 25
mL of CH2Cl2, and the combined organics were dried over MgSO4,
filtered, and concentrated. The crude oil was purified by flash
chromatography (20% to 30% EtOAc/Hexanes) to give 3.106 g
(95.9%) of a pale yellow oil that slowly solidified. 1H NMR (CDCl3,
399.5 MHz) δ 7.32-7.16 (m, 3H), 7.14-7.06 (m, 3H), 6.88-6.80
(m, 2H), 6.73 (d, J ) 7.9 Hz, 1H), 4.73 (d, J ) 7.6 Hz, 1H), 4.71
(d, J ) 7.9 Hz, 1H), 4.50 (ABq, νA ) 31.7, νB ) 11.5 Hz, 2H),
4.42-4.33 (m, 1H), 3.99 (dd, J ) 9.0, 2.0 Hz, 1H), 3.96 (dd, J )
6.9, 3.9 Hz, 1H), 3.90-3.82 (m, 2H), 3.82 (s, 3H), 3.78 (s, 3H),
3.23 (dd, J ) 13.3, 3.1 Hz, 1H), 2.67 (dd, J ) 13.3, 9.9 Hz, 1H),
1.63 (qd, J ) 7.7, 4.6 Hz, 2H), 1.23 (d, J ) 7.0 Hz, 3H), 0.96 (t,
J ) 7.5 Hz, 3H); 13C NMR (CDCl3, 100.5 MHz) δ 10.1, 10.8,
25.6, 37.6, 41.2, 55.8, 66.0, 69.8, 80.1, 94.3, 110.7, 110.8, 119.9,
127.2, 128.8, 129.3, 130.5, 135.4, 148.5, 148.9, 153.3, 174.9; HRMS
ESI+ (m/z): 494.2160 (Predicted [M + Na]+ for C26H33NO7 is
494.2155).
Amide 5. n-BuLi (2.35 M in hexanes, 33.58 mL, 78.91 mmol)
was added dropwise at -78 °C to a stirring suspension of flame-
dried LiCl (12.948 g, 305.45 mmol) and diisopropylamine (11.871
mL, 84.00 mmol) in 50 mL of THF. The reaction was warmed to
0 °C briefly for 5 min and then recooled to -78 °C. (S,S)-(+)-
pseudoephedrine propionamide (9.013 g, 40.73 mmol) in 100 mL
THF was added dropwise by cannula, and the reaction was stirred
for 1 h at -78 °C, 30 min at 0 °C, and 5 min at room temperature.
Iodide 4 (8.000 g, 25.45 mmol) in 10 mL of THF was added
dropwise at 0 °C, and then the reaction was allowed to warm to
room temperature and stirred for 22 h. The reaction was quenched
with 100 mL of saturated NH4Cl and diluted with 100 mL of
EtOAc. The layers were separated, and the aqueous layer was
extracted with 2 × 100 mL EtOAc. The combined organics were
dried over MgSO4, filtered, and concentrated. The crude oil was
purified by flash chromatography (30% EtOAc/Hexanes) to give
8.994 g (86.7%) of a colorless solid. 1H NMR (CDCl3, 399.5 MHz)
δ 7.43-7.10 (m, 5H), 4.64-4.51 (m, 2H), 4.34 (s, 1H), 3.43 (dd,
J ) 9.8, 4.8 Hz, 1H), 3.36 (dd, J ) 9.8, 5.9 Hz, 1H), 2.83 (s, 3H),
2.80-2.64 (m, 1H), 1.70-1.60 (m, 1H), 1.58-1.47 (m, 1H), 1.13
(d, J ) 7.1 Hz, 3H), 1.20-1.04 (m, 1H), 1.07 (d, J ) 6.7 Hz, 3H),
0.86 (s, 9H), 0.81 (d, J ) 6.6 Hz, 3H), 0.01 (s, 6H); 13C NMR
(CDCl3, 100.5 MHz) δ -5.5, -5.4, 14.4, 17.3, 17.5, 18.3, 25.9,
33.1, 34.1, 37.6, 67.9, 76.5, 76.7, 77.0, 77.3, 126.2, 126.9, 127.4,
128.3, 128.7, 142.6, 179.1; HRMS ESI+ (m/z): 430.2740 (Predicted
[M + Na]+ for C23H41NO3Si is 430.2753). As expected from
literature precedent,19 5 was isolated as a mixture of amide rotamers.
The reported NMR data only denote peaks arising from the major
rotamer.
Alcohol 9c. LiBH4 (2.0 M in THF, 3.83 mL, 7.66 mmol) was
added dropwise to a solution of oxazolidinone 8 (3.010 g, 6.38
mmol) and MeOH (0.341 mL, 7.66 mmol) in 30 mL of MTBE at
0 °C. The reaction was stirred for 20 min, then warmed to room
temperature, and monitored by TLC. After 3 h, the reaction was
quenched with 30 mL of saturated Na/K tartrate and stirred until
the layers became clear. The layers were separated, and the aqueous
portion was extracted with 2 × 25 mL of EtOAc. The combined
organics were dried over MgSO4, filtered, and concentrated. The
crude oil was purified by flash chromatography (30% EtOAc/
Ketone 6. To a stirring suspension of amide 5 (1.866 g, 4.58
mmol) in 50 mL of THF was added EtLi (0.46 M in 90/10 benzene/
cyclohexane, 22.89 mL, 10.53 mmol) at -78 °C. The reaction was
stirred for 10 min, then warmed to 0 °C, and stirred an additional
30 min. Subsequently, 1 equiv of diisopropylamine was added to
scavenge excess EtLi, and the mixture was stirred for 15 min. The
reaction was quenched with 20% AcOH/Et2O and diluted with H2O
and EtOAc, and the layers were separated. The aqueous portion
was extracted with 1 × 50 mL EtOAc, and the combined organics
were dried over MgSO4, filtered, and concentrated. The crude oil
was purified by flash chromatography (2% EtOAc/Hexanes) to give
1
Hexanes) to give 1.716 g (90.1%) of a pale yellow oil. H NMR
(CDCl3, 399.5 MHz) δ 6.91-6.75 (m, 3H), 4.75 (ABq, νA ) 17.4,
νB ) 6.9 Hz, 2H), 4.55 (ABq, νA ) 31.9, νB ) 11.5 Hz, 2H), 3.85
(s, 3H), 3.83 (s, 3H), 3.71-3.58 (m, 1H), 3.58-3.39 (m, 1H), 2.69
(br s, 1H), 2.06-1.86 (m, 1H), 1.68-1.54 (m, 1H), 1.54-1.41 (m,
0H), 0.90 (t, J ) 7.4 Hz, 3H), 0.82 (d, J ) 7.0 Hz, 3H); 13C NMR
(CDCl3, 100.5 MHz) δ 10.5, 10.8, 24.1, 37.6, 55.8, 55.9, 65.3, 69.9,
81.2, 94.3, 110.9, 111.1, 120.4, 130.0, 148.7, 149.0; HRMS ESI+
(m/z): 321.1672 (Predicted [M + Na]+ for C16H26O5 is 321.1678).
Aldehyde 3c. To a suspension of alcohol 9 (1.251 g, 4.19 mmol)
and NaHCO3 (1.761 g, 20.96 mmol) in 40 mL of CH2Cl2 was added
Dess-Martin periodinane (2.133 g, 5.03 mmol) at 0 °C. The reaction
was stirred for 5 min, then warmed to room temperature, and
monitored by TLC. After 1 h, the reaction was quenched by addition
of 20 mL of half saturated Na2S2O3 and stirred until the layers
became clear. The layers were separated, and the aqueous portion
was extracted with 2 × 25 mL of CH2Cl2. The combined organics
were dried over MgSO4, filtered, and concentrated. The crude oil
was purified by flash chromatography (20% EtOAc/Hexanes) to
give 0.941 g (75.7%) of a pale yellow oil. 1H NMR (CDCl3, 399.5
MHz) δ 9.78 (s, 1H), 7.10-6.41 (m, 1H), 4.75 (ABq, νA )18.1,
νB ) 7.2 Hz, 2H), 4.47 (ABq, νA ) νB ) 11.4 Hz, 2H), 4.01 (td,
J ) 6.8, 3.4 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 2.56 (qd, J ) 7.0,
3.4 Hz, 1H), 1.73-1.64 (m, 1H), 1.63-1.50 (m, 1H), 1.11 (d, J )
7.0 Hz, 3H), 0.94 (t, J ) 7.4 Hz, 3H); 13C NMR (CDCl3, 100.5
MHz) δ 7.6, 10.3, 24.8, 49.4, 55.8, 55.9, 69.7, 78.6, 93.8, 110.9,
1
1.101 g (88.3%) of a colorless oil. H NMR (CDCl3, 399.5 MHz)
δ 3.39 (dd, J ) 10.3, 6.4 Hz, 1H), 3.35 (dd, J ) 10.2, 6.5 Hz, 1H),
2.70-2.60 (m, 1H), 2.52-2.35 (m, 2H), 1.77 (ddd, J ) 13.8, 7.9,
6.1 Hz, 1H), 1.60-1.44 (m, 2H), 1.06 (d, J ) 6.9 Hz, 3H), 1.02 (t,
J ) 7.3 Hz, 3H), 0.87 (s, 9H), 0.86 (d, J ) 6.4 Hz, 3H), 0.01 (s,
6H); 13C NMR (CDCl3, 100.5 MHz) δ -5.5, -5.5, 7.7, 17.1, 17.3,
18.2, 25.8, 33.6, 33.6, 36.9, 43.9, 67.9, 215.2; HRMS ESI+ (m/z):
295.2076 (Predicted [M + Na]+ for C15H32O2Si is 295.2069).
Keto Acid 2. Ketone 6 (1.081 g, 3.97 mmol) was dissolved in
20 mL of 1:1:2 CCl4/CH3CN/H2O, and then RuCl3 ·H2O (0.082 g,
0.397 mmol) and NaIO4 (4.242 g, 19.84 mmol) were added
sequentially. The reaction was brought to reflux at 70 °C and heated
for 16 h overnight. The mixture was cooled to room temperature,
diluted with 20 mL of CH3CN, and filtered through a plug of Celite.
The plug was washed with an additional 100 mL of CH3CN, and
the filtrate volatiles were removed en Vacuo. The mixture was taken
up in 50 mL of EtOAc and extracted with 3 × 50 mL of half
saturated NaHCO3. The combined aqueous portion was then back
extracted with 1 × 50 mL Et2O. The aqueous layer was then
carefully acidified with dropwise addition of concentrated HCl to
pH 2 and then extracted with 4 × 50 mL CH2Cl2. The organic
extracts were dried over MgSO4, filtered, and concentrated to give
(35) Prepared as previously described: Trost, B. M.; Frederiksen, M. U.;
Papillon, J. P. N.; Harrington, P. E.; Shin, S.; Shireman, B. T. J. Am.
Chem. Soc. 2005, 127, 3666–3667.
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Erythromycin and Pikromycin Polyketide Synthases
A R T I C L E S
111.3, 120.5, 130.1, 148.7, 149.0, 204.5; HRMS ESI+ (m/z):
319.1521 (Predicted [M + Na]+ for C16H24O5 is 319.1527).
DMBOM-Protected DEBS Pentaketide SNAC 11. LiHMDS
(1.0 M in THF, 4.85 mL, 4.85 mmol) was added dropwise to a
stirring solution of keto acid 2 (0.348 g, 2.03 mmol) and flame-
dried LiCl (0.343 g, 8.10 mmol) in 10 mL of THF at -78 °C. The
reaction was stirred for 1 h and then warmed to 0 °C for 30 min.
The reaction was then recooled to -78 °C, and the aldehyde 3c
(0.901 g, 3.04 mmol) in 5 mL of THF was added dropwise.
Following addition, the reaction was stirred for 90 min and then
quenched with 20 mL of saturated NH4Cl. The mixture was diluted
with 20 mL of EtOAc and then extracted with 3 × 25 mL of half
saturated NaHCO3. The aqueous extracts were back extracted with
1 × 25 mL of Et2O and then carefully acidified to pH 2 with 1 N
HCl. The aqueous layer was then extracted with 4 × 25 mL of
CH2Cl2. The combined organics were dried over MgSO4, filtered,
and concentrated. The crude oil (containing a ∼7:1 anti-Felkin/
148.6, 149.0, 171.4, 203.7, 217.7; HRMS ESI+ (m/z): 592.2933
(Predicted [M + Na]+ for C29H47NO8S is 592.2920).
DEBS Pentaketide SNAC 1. DDQ (0.018 g, 0.079 mmol) was
added to 11 (0.009 g, 0.016. mmol) in a biphasic mixture of 4:1
CH2Cl2/0.5 M phosphate buffer, pH 7.0 at 0 °C. The reaction was
warmed to room temperature and monitored by TLC. The reaction
was quenched with 5 mL of saturated NaHCO3 and diluted with
10 mL of CH2Cl2. The layers were separated, and the aqueous
portion was extracted with 2 × 10 mL of CH2Cl2. The combined
organics were dried over MgSO4, filtered, and concentrated. The
crude material was resuspended in 1 mL of CH3CN, run through a
0.22 µm hydrophobic syringe filter (Millipore), and then purified
by preparative HPLC on a Phenomenex Luna C18(2) column (5
µm, 21.2 mm × 250 mm) using a CH3CN/H2O gradient (10% to
100%, 60 min, 10 mL/min). The desired compound eluted at 33
and 37.5 min (detecting at 240 nm) to give 2.4 mg (39%) of a
colorless oil as an ∼2:1 equilibrium mixture of open linear
1
1
pentaketide and closed hemiketal. H NMR (CDCl3, 399.5 MHz)
Felkin mixture of seco-acids by H NMR) was carried on to the
δ 5.91 (br s, 0.35H), 5.85 (br s, 0.65H), 3.91 (dt, J ) 8.8, 2.8 Hz,
0.65H), 3.87-3.73 (m, 1.35H), 3.58-3.27 (m, 3H), 3.10-2.97 (m,
2H), 2.85 (ddd, J ) 14.2, 7.1, 2.9 Hz, 1H), 2.81-2.61 (m, 2H),
2.12 (ddd, J ) 14.4, 8.7, 5.8 Hz, 1H), 1.97 (s, 2H), 1.96 (s, 1H),
1.90-1.76 (m, 2H), 1.73 (ddd, J ) 9.0, 7.0, 2.0 Hz, 0.65H),
1.64-1.50 (m, 1.65H), 1.48-1.31 (m, 2H), 1.23 (d, J ) 7.0 Hz,
1H), 1.20 (d, J ) 6.9 Hz, 2H), 1.14 (d, J ) 7.2 Hz, 2H), 1.11 (d,
J ) 6.9 Hz, 2H), 0.98 (t, J ) 7.4 Hz, 2H), 0.96 (d, J ) 5.8 Hz,
1H), 0.95 (d, J ) 6.5 Hz, 1H), 0.89 (t, J ) 7.4 Hz, 1H), 0.84 (d,
J ) 7.0 Hz, 2H), 0.82 (d, J ) 6.7 Hz, 1H); 13C NMR (CDCl3,
100.5 MHz) δ 4.5, 9.5, 10.4, 11.0, 11.0, 15.0, 16.3, 16.4, 18.6,
19.8, 23.2, 25.2, 26.4, 28.4, 28.5, 29.7, 34.7, 35.9, 36.3, 37.9, 38.9,
42.7, 45.6, 46.3, 47.4, 71.8, 73.0, 73.6, 74.4, 76.7, 100.4, 170.4,
203.6, 218.5; HRMS ESI+ (m/z): 412.2119 (Predicted [M + Na]+
for C19H35NO5S is 412.2134).
next step without further purification.
The crude acid was dissolved in 20 mL of CH2Cl2, followed by
addition of HATU (1.152 g, 3.03 mmol), DIPEA (1.06 mL, 6.06
mmol), and HSNAC (0.258 mL, 2.43 mmol). The reaction became
clear, and the dark yellow solution was stirred for 18 h at room
temperature. The reaction was quenched with 20 mL of saturated
NaHCO3, and the resulting layers were separated. The aqueous layer
was then extracted with 2 × 25 mL of CH2Cl2. The combined
organics were dried over MgSO4, filtered, and concentrated. The
crude oil was partially purified by flash chromatography (2%
MeOH/CH2Cl2) and then purified by preparative HPLC on a
Phenomenex Luna C18(2) column (5 µm, 21.2 mm × 250 mm)
using a CH3CN/H2O + 0.1% TFA gradient (10% to 100%, 60 min,
10 mL/min). The desired compound eluted between 45.5 and 46.5
(detecting at 254 nm) min to give 0.168 g (14.6%) of a colorless
oil. 1H NMR (CDCl3, 399.5 MHz) δ 6.90-6.66 (m, 1H), 6.05 (br
s, 1H), 4.78 (ABq, νA ) 23.0, νB ) 6.8 Hz, 2H), 4.55 (ABq, νA )
νB ) 11.5 Hz, 2H), 3.95 (dd, J ) 9.5, 2.5 Hz, 1H), 3.89-3.85 (m,
1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.51 - 3.30 (m, 2H), 3.10-2.89
(m, 2H), 2.82-2.73 (m, 2H), 2.69-2.62 (m, 1H), 2.09 (ddd, J )
14.5, 8.6, 6.2 Hz, 1H), 1.97 (s, 3H), 1.82-1.65 (m, 2H), 1.56-1.44
(m, 1H), 1.37-1.27 (m, 1H), 1.16 (d, J ) 6.8 Hz, 3H), 1.08 (d, J
) 7.0 Hz, 3H), 1.06 (d, J ) 6.8 Hz, 3H), 0.91 (t, J ) 7.4 Hz, 3H),
0.83 (d, J ) 6.8 Hz, 3H); 13C NMR (CDCl3, 100.5 MHz) δ 8.6,
10.3, 10.7, 16.8, 18.6, 22.8, 24.6, 28.2, 36.8, 37.7, 39.8, 42.0, 46.4,
46.4, 55.8, 55.9, 69.8, 72.0, 80.6, 94.6, 110.9, 111.1, 120.4, 130.0,
Acknowledgment. This research was supported by a Rackham
Regents’ Predoctoral Fellowship and a graduate research fellowship
from Eli Lilly & Co. (to J.D.M.), an NIH NRSA (GM075641)
Postdoctoral Fellowship (to J.D.K), and NIH Grant GM076477 and
the Hans W. Vahlteich Professorship (to D.H.S.).
Supporting Information Available: Experimental details. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JA9060596
9
J. AM. CHEM. SOC. VOL. 131, NO. 43, 2009 15793